Targeting of podoplanin with lectin for use in the prevention and treatment of cancer

ABSTRACT

Lectin compositions and methods for reducing tumor cell growth and preventing or treating cancer are provided.

INTRODUCTION

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/401,849, filed Mar. 11, 2009, which claimsbenefit of priority from U.S. Provisional Patent Application Ser. No.61/069,198, filed Mar. 12, 2008, and 61/085,039, filed Jul. 31, 2008,the contents of each of which are incorporated herein by reference intheir entirety.

This invention was made with government support under grant numberCA88805-01A2 and R01CA126897 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Progress in methods to detect and treat cancer has led to increasedfive-year survival rates. However, cancer mortality rates have notdecreased proportionately (Welch, et al. (2000) JAMA 283:2975-2978).Some transformed cells can survive medical treatment and lay dormant foryears before they emerge to cause relapse in a patient. Nontransformedcells can force tumor cells to assume a normal morphology and phenotypeby a process called “Contact Normalization” (Glick & Yuspa (2005) Semin.Cancer Biol. 15:75-83; Rubin (2003) Adv. Cancer Res. 90:1-62).

Increased anchorage-independent growth and migration distinguish cancercells from their nontransformed precursors (Frisch & Screaton (2001)Curr. Opin. Cell Biol. 13:555-562; Giancotti & Buoslahti (1999) Science285:1028-1032). Src kinase, a membrane-bound tyrosine kinase,phosphorylates Cas (Crk associated substrate) to promote thesefundamental hallmarks of tumor cell growth (Brabek, et al. (2004)Oncogene 23:7406-7415; Cho & Klemke (2000) J. Cell Biol. 149:223-236;Goldberg, et al. (2003) J. Biol. Chem. 278:46533-46540; Honda, et al.(1998) Nat. Genet. 19:361-365; Huang, et al. (2002) J. Biol. Chem.277:27265-27272; Klemke, et al. (1998) J. Cell Biol. 140:961-972; Shin,et al. (2004) J. Biol. Chem. 279:38331-38337).

Src has been implicated in many human cancers (Frame (2002) Biochim.Biophys. Acta 1602:114-130; Irby & Yeatman (2000) Oncogene19:5636-5642). Increased Src activity has been reported in cancers ofthe colon (Cartwright, et al. (1994) J. Clin. Invest 93:509-515;Cartwright, et al. (1989) J. Clin. Invest 83:2025-2033; Cartwright, etal. (1990) Proc. Natl. Acad. Sci. USA 87:558-562), and particularly inmetastatic colon cancer that invades liver (Termuhlen, et al. (1993) J.Surg. Res. 54:293-298). Increased Src activity is also found in cancersof the pancreas (Lutz, et al. (1998) Biochem. Biophys. Res. Commun.243:503-508), lung (Mazurenko, et al. (1992) Eur. J. Cancer 28:372-377);neural cells (Bjelfman, et al. (1990) Mol. Cell Biol. 10:361-370; Bolen,et al. (1985) Proc. Natl. Acad. Sci. USA 82:7275-7279), muscle (Rosen,et al. (1986) J. Biol. Chem. 261:13754-13759), ovaries (Budde, et al.(1994) Cancer Biochem. Biophys. 14:171-175; Wiener, et al. (1999) Clin.Cancer Res. 5:2164-2170), esophagus (Kumble, et al. (1997)Gastroenterology 112:348-356), skin (Bjorge, et al. (1996) Biochem. CellBiol. 74:477-484; Munshi, et al. (2000) J. Immunol. 164:1169-1174),stomach (Takeshima, et al. (1991) Jpn. J. Cancer Res. 82:1428-1435), andbreast (Ottenhoff-Kalff, et al. (1992) Cancer Res. 52:4773-4778; Rosen,et al. (1986) supra; Verbeek, et al. (1996) J. Pathol. 180:383-388).Indeed, metastatic cell growth can be reduced by agents that inhibit Srcactivity (Lutz, et al. (1998) supra; Roginskaya, et al. (1999) Leukemia13:855-861; Staley, et al. (1997) Cell Growth Differ. 8:269-274).

Cas is an important component of the focal adhesion complex signalingnetwork (Bouton, et al. (2001) Oncogene 20:6448-6458) that also includesFAK, Grb2, Shc, and paxillin (Schlaepfer, et al. (1999) Prog. Biophys.Mol. Biol. 71:435-478; Sieg, et al. (1999) J. Cell Sci. 112 (Pt16):2677-2691). After phosphorylation by Src, Cas an bind to otherproteins including Crk, PI-3-kinase, Nc, and PLCy (Burnham, et al.(1996) Oncogene 12:2467-2472; Sakai, et al. (1994) EMBO J. 13:3748-3756;Vuori, et al. (1996) Mol. Cell Biol. 16:2606-2613). Src transformationof homozygous null Cas knockout (CasKO) cells does not fully promotetheir anchorage-independence or ability to migrate. These transformedgrowth characteristics can be conferred to CasKO cells by transfectionwith wild-type Cas (Brabek, et al. (2005) Mol. Cancer Res. 3:307-315;Goldberg, et al. (2003) supra; Honda, et al. (1998) supra; Huang, et al.(2002) supra).

Src phosphosphorylates Cas to inhibit Fh11 expression thereby promotingnonanchored cell growth and migration (Shen, et al. (2006) Cancer Res.66:1543-1552). Fhll is composed of four and half LIM domains and canmove between intercellular junctions (Huang, et al. (2002) Yi. ChuanXue. Bao. 29:953-958), focal adhesions, and the nucleus (Brown, et al.(1999) J. Biol. Chem. 274:27083-27091) to affect gene expression (Qin,et al. (2005) FEBS Lett. 579:1220-1226; Taniguchi, et al. (1998) Mol.Cell Biol. 18:644-654). Transfection studies demonstrate that Fh11specifically blocks nonanchored tumor cell growth and migration, butdoes not affect “normal” anchored cell growth (Shen, et al. (2006)supra). Therefore, Fhll acts as a true tumor suppressor rather than as ageneral mitotic inhibitor. Analysis of human clinical specimens suggeststhat Fhll expression is suppressed in some human tumors including thoseof breast, kidney, and prostate. Furthermore, Fhll expression issuppressed by Src, and is restored during Contact Normalization oftransformed cells (Shen, et al. (2006) supra). Thus, Fhll actsdownstream of Src and Cas to suppress the anchorage-independence andmotility that leads to metastatic cell growth.

Lectin, which is a protein selectively binding to carbohydrates, hasoriginally been discovered from plants, microorganisms, virus,invertebrates or vertebrates and widely used as a material for variousdisease-related researches. Lectin has various functions: it has beenknown to agglutinate erythrocytes to sediment them, and has been used indetermining blood type. In addition, it has been shown to have afunction of specifically agglutinating cancer cells and lectins fromKorean legume Maackia fauriei and Korean marine crab Philyra pisum havebeen shown to exhibit anti-proliferative activity (U.S. Pat. No.7,045,300 and U.S. Pat. No. 7,015,313).

SUMMARY OF THE INVENTION

The present invention features methods for preventing or reducing tumorcell growth and migration, reducing tumor size and vascularization andpreventing or treating cancer with an isolated lectin that binds sialicacid. In some embodiments, the lectin is administered orally. In otherembodiments, the lectin is a plant lectin from Maackia amurensis,Trichosanthes japonica, Viscum album, Artocarpus intergrifolia, orSambucus nigra. In particular embodiments the lectin has the sequenceset forth in SEQ ID NO:23. Pharmaceutical compositions and kits are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a layered culture system. Nontransformed or Srctransformed Cx43Ko cells were incubated beneath or on top of a porousmembrane that prevents actual mixing of the cell populations.Transformed cells separated by 1 mm from nontransformed cells enabledcommunication by diffusible factors but not intercellular junctions.Transformed cells incubated directly over nontransformed cells allowedfor intercellular junctional communication and Contact Normalization.

FIGS. 2A and 2B show the effect of lectins in a wound healing migrationassay (FIG. 2A) and toxicity assay (FIG. 2B). Migration assays wereperformed on confluent monolayers of cells treated with varyingconcentrations of Maackia amurensis lectin (MAA) as indicated. Data areshown as the number of cells that migrated into a 300×300 micron areaalong the center of the wound in 24 hours (mean+SEM, n=7). MAA toxicitywas evaluated by Trypan blue staining of cells from wound healingassays. Data are shown as thepercent of live cells from equivalentsamples of each well (mean+SEM, n=4).

DETAILED DESCRIPTION OF THE INVENTION

A comprehensive analysis has now been performed to identify genes thatare modulated during Contact Normalization. Contact normalizationdescribes the ability of nontransformed cells (i.e., normal cells) tonormalize the growth of neighboring transformed or cancer cells. Tumorcells need to overcome this form of growth inhibition before they canbecome malignant or metastatic. The analysis herein identified specificgenes which were activated in transformed cells, but suppressed innontransformed cells. Moreover, the expression of these genes wasinhibited in transformed cells that were undergoing ContactNormalization. In addition, genes whose expression was decreased intransformed cells, but increased by Contact Normalization were alsoidentified. Thus, these genes serve as viable biomarkers andchemotherapeutic targets in the diagnosis, prognosis and treatment ofcancer. Indeed, analysis of one particular biomarker, Podoplanin (Pdpn;GENBANK Accession Nos. NP_001006625, NP_001006626, NP_006465, andNP_938203), indicated that targeting this unique transmembrane receptorwith a lectin that binds sialic acid, inhibits transformed cell growthand migration in vitro. In particular, it was shown that M. amurensislectin inhibits cell migration prior to inhibiting cell viability. Forexample, 308 nM MAA suppresses melanoma cell migration by over 99%,while inhibiting cell viability by about 20% within the same timeperiod. In addition, dietary administration of lectin inhibitedsubcutaneous melanoma growth and vascularization in mice. This analysisdemonstrated that lectins can be used to target Pdpn to attack cancercells.

Accordingly, the present invention provides the use of a lectin thatbinds sialic acid on Pdpn in methods for preventing or reducing tumorcell growth and migration, reducing tumor size and vascularization andpreventing or treating cancer. In some embodiments, the lectin isisolated and optionally purified. As used herein, a protein is said tobe “isolated” when physical, mechanical or chemical methods are employedto remove the protein from cellular constituents that are normallyassociated with the protein. A skilled artisan can readily employstandard purification methods to obtain an isolated protein. A purifiedlectin molecule will be substantially free of other proteins ormolecules and is generally 95% to 100% homogenous. The nature and degreeof isolation and purification will depend on the intended use.

The lectin of the present invention can be isolated and optionallypurified using conventional methods. For example, when isolated from itsnatural source, the lectin can be purified to homogeneity on appropriateimmobilized carbohydrate matrices and eluted by proper haptens. See,Goldstein & Poretz (1986) In The lectins. Properties, functions andapplications in biology and medicine (ed. Liener et al.), pp. 33-247.Academic Press, Orlando, Fla.; Rudiger (1993) In Glycosciences: Statusand perspectives (ed. Gabius & Gabius), pp. 415-438. Chapman and Hall,Weinheim, Germany. Alternatively, the lectin can be produced byrecombinant methods according to established methods. See Streicher &Sharon (2003) Methods Enzymol. 363:47-77. As yet another alternative,lectins can be generated using standard peptide synthesis technology orusing chemical cleavage methods well-known in the art based on the aminoacid sequences of known lectins or the lectin disclosed herein.

The lectin can also be modified in a way to form a chimeric moleculecontaining lectin fused to another, heterologous polypeptide or aminoacid sequence. In one embodiment, such a chimeric molecule is a fusionof the lectin with a polyhistidine epitope tag, which provides anepitope to which immobilized nickel can selectively bind. The epitopetag is generally placed at the amino- or carboxyl-terminus of a protein.In an alternative embodiment, the chimeric molecule can include a fusionof the lectin with an immunoglobulin or a particular region of animmunoglobulin.

Lectins of particular use in the instant invention are those that bindsialic acid. Examples of such lectins include those listed in Table 1.

TABLE 1 Species or Organism Cell Type Lectin Binding Specificity VirusSendai virus HN NeuAcα2, 3Galpβ1, 3GalNac/4GlcNAc Human HN NeuAcα2,3Galβ1, 4GlcNac parainfluenza virus type 1 Bacteria Streptococcus SABPNeu5Acα2, 3Galβ1, 3GalNac mitis Mycoplasma HA-A Neu5Acα2, 3Galβ1,4GloNAcP pneumoniae 1,3 Fungi Polyporus PSA Neu5Acα2, 6Galβ1, 4G1c/Glcsquamosus NAc Psathyrella PVS Neu5Acα2, 6Galβ1, 4G1cNAc vetutinaMacrophomina MPL Neu5Acα2, 3Galβ1, 4G1cNAc phaseolina Plants Maackia MALNeu5Acα2, 3Galβ1, 4G1cNAc amurensis MAH Neu5Acα2, 3Galβ1, 3 [Neu5Ac a2,6] GalNAc Trichosanthes TJA Neu5Acα2, 6Galβ1, 4G1cNAc japonica Viscumalbum ML-1 Neu5Acα2, 6Galβ1, 4G1cNAc Artocarpus jacalin Gal and Man >Neu5Ac intergrifolia Sambucus SNA Neu5Acα2, −6Ga1 (NAc) -R nigra InsectsAllomyrina Allo A-II Neu5Acα2, 6Ga1β1, 4G1cNAc dichotoma Animals bloodIL-lα Neu5Acα2, 3Galβ1, 4G1cNAc macrophages Siglec-1 Neu5Acα, 3Gal

For example, the M. amurensis lectins are composed of two types oflectin molecules (MAH and MAL), which display slightly different bindingpreferences. It has been shown that this mixture can specificallysuppress anchorage-independence and motility of Src transformed cells atnM concentrations. Analysis of MAH and MAL separately can determine ifeither of these compounds is more effective at preventing malignant cellgrowth than the mixture found in nature.

As shown in Table 1, sialic acid (N-acetylneuraminic acid; Neu5Ac orNANA) binding lectins can be derived from virus, bacterial, fungal,plant, insect, and animal sources. For example, analysis hereinindicates that jacalin, which preferentially associates withglycoconjugates containing galactosyl (β1,3)N-acetylgalactosamine, mayactually augment tumor cell migration. In contrast, MAH or MAL, whichpreferentially associate with 0-glycosidically linked oligosaccharideshaving galactosyl (β1,4)N-acetylglucosamine structures with (α2,3)linked sialic acid, can prevent nonanchored tumor cell growth andmigration. Taken together, this analysis indicates that specific lectinscan associate with Pdpn and suppress nonanchored cell growth andmigration required for tumor cell invasion and progression. In thisrespect, lectins such as MPL from M. phaseolina and TJA from T. japonicacan be analyzed for similar binding specificities. These compounds canbe obtained from a variety of sources including Sigma-Aldrich,Calbiochem, and Vector Laboratories. In particular embodiments, thelectin used in accordance with the present invention is that set forthin SEQ ID NO:23.

Fluorescence microscopy can be used to determine if specific lectinscolocalize with Pdpn. Moreover, immunoprecipitation can be used todetermine if Pdpn and lectins associate with each other. To determine iflectins that bind to Pdpn can suppress nonanchored growth and migrationof Src transformed cells, Src transformed cells and nontransformed cellsare treated with various concentrations of specific lectins that bind toPdpn, as well other lectins with different binding affinities (see,e.g., Table 1). Mouse embryonic fibroblasts, and other established celllines, including LA25 cells, can be used for these experiments. Theeffects of several concentrations of specific lectins on the anchoredgrowth, nonanchored growth, and migration of Src transformed andnontransformed cells are quantitated. It is expected that specificlectins will inhibit the malignant growth potential of tumor cells atconcentration that will not inhibit the “normal” growth ofnontransformed cells. This is demonstrated by showing that specificlectins significantly inhibit nonanchored growth and migration oftransformed cells, without significantly interfering with the anchoredgrowth of parental nontransformed control cells.

For use in vitro or an experimental setting, e.g., to identifysynergistic therapies in a cell-based or animal model system, a tumorcell is contacted with a lectin that binds sialic acid so that tumorcell growth and migration is measurably decreased, reduced or inhibited,e.g., by 25%, 50%, or 75% as compared to a tumor cell not contacted withthe lectin. The term “contacted” when applied to a cell is used hereinto describe the process by which a lectin is delivered to a target cellor is placed in direct proximity with the target cell. This delivery maybe in vitro or in vivo and may involve the use of a recombinant vectorsystem. Any method may be used to contact a cell with lectin, so long asthe method results in either increased levels of lectin at the tumorcell. This includes both the direct delivery of lectin to the cell andthe delivery of a gene or DNA segment that encodes the lectin.

Lectins that bind to sialic acid can also be used in therapeuticapproaches for the prevention or treatment of cancers includingmalignant and metastatic cancers in subjects with cancer, subjects atrisk of developing cancer or subjects harboring cancer cells affected byContact Normalization. For therapeutic applications, the lectin isadministered, e.g., in a pharmaceutically acceptable carrier and/orappropriate delivery vehicle, to reduce tumor size and vascularizationand/or treat cancer. As used herein, the term “tumor” means a mass oftransformed cells that are characterized, at least in part, bycontaining angiogenic vasculature. Although a tumor generally is amalignant tumor, i.e., a cancer having the ability to metastasize, atumor also can be nonmalignant, provided that neovascularization isassociated with the tumor. In some embodiments, tumor cells contemplatedherein are derived from metastatic tumors. As used herein, the term“metastasis” refers to a secondary tumor that grows separately from theprimary and has arisen from detached, transported cells.

The methods described herein are contemplated for use in the preventionor treatment of cancer and the incidence of tumor metastasis for allforms of tumors and cancer. Exemplary forms of cancer contemplatedherein for treatment include head and neck squamous cell carcinoma,colon carcinoma, renal cancer, lung adenocarcinoma, human gastriccancer, breast cancer, cancer of the central nervous system, pancreaticcancer, liver cancer, ovarian cancer, prostate cancer, leukemia, skincancer and the like. In particular embodiments, lectin is used in thetreatment of a mammary carcinoma or melanoma.

As shown in the Examples, an effective amount of lectin inhibitedtransformed cancerous cells from proliferating and migrating and fromforming a tumor in vivo. In view of the results disclosed herein, theskilled artisan would recognize that an effective amount of a particularlectin can vary depending on several factors, including the activity ofthe lectin, other components, if any, present in a pharmacologicallyacceptable composition containing the lectin, and the physico-chemicalproperties of the lectin. In accordance with the present invention, an“effective amount” of lectin is an amount that binds to Pdpn in asubject and inhibits tumor cell proliferation, vascularization and/ormigration. In particular embodiments, treatment with a lectin disclosedherein provides at least a 25, 50, or 75% in tumor size as well as ameasurable decrease in vascularization. In this respect, therapeutic useof the instant lectins can include the additional step of determiningwhether the tumor cells of the subject (e.g., cells of a biopsy sample)will bind lectin via Pdpn prior to administration and/or whether thelectin binds to Pdpn after administration.

As used herein, a “subject” means a mammal, including, for example, ahuman, a monkey or a cat, and the like. Subjects benefiting fromtherapeutic treatment with lectin include subjects wherein cancer hasalready been identified or has started progressing or metastasizing.Subjects benefiting from prophylactic treatment with lectin includesubjects with a predisposition or increased risk of cancer (e.g.,because of genetics or exposure to carcinogens), wherein cancer has notyet formed, established, progressed or metastasized.

An effective amount of lectin is preferably within the range of about0.01 to about 500 mg/kg body weight, and can be readily determined byconsidering the activity of the particular lectin being administered,the route of administration, the period over which the lectin is to beadministered, and other factors known to those skilled in the art. Thus,the lectins described herein can be used as medicaments for thetreatment of a variety of cancer pathologies by inhibiting tumor cellgrowth and metastasis.

The in vivo effect of a therapeutic composition may be evaluated in asuitable animal model. For example, xenogenic cancer models whereinhuman cancer explants or passaged xenograft tissues are introduced intoimmune compromised animals, such as nude or SCID mice, are appropriatein relation to cancer and have been described (Klein, et al. (1997)Nature Medicine 3:402-408). For example, WO 98/16628 describes variousxenograft models of human prostate cancer capable of recapitulating thedevelopment of primary tumors, micrometastasis, and the formation ofosteoblastic metastases characteristic of late stage disease. Efficacymay be predicted using assays that measure inhibition of tumorformation, tumor regression or metastasis, and the like.

The lectin preparation, together with conventional additives, carriersor diluting agents, can be used to prepare pharmaceutical compositions,including individual doses thereof, in the form of tablets, filledcapsules, or fluids such as solutions, mixtures, emulsions, elixirs orcapsules filled with such, all for oral intake, as well as in the formof suppositories. Such pharmaceutical compositions and individual dosesthereof can include conventional ingredients or principles, and suchdosage-forms can contain any effective concentration of the activeingredients in accordance with the intended daily dosage range.Preparations that contain approximately 100, 200 or 300 mg of the lectinper individual dosage unit are representative of an appropriateconcentration. However, dosages and administration protocols for thetreatment of cancers using the foregoing methods may vary with themethod and the target cancer and will generally depend on a number ofother factors appreciated in the art.

The pharmaceutical composition according to the invention can beadministered in a wide range of dosage-forms. Carriers used to produce apharmaceutical containing the instant lectin can include both solid andliquid substances. Solid dosage-forms may include powders, tablets,pills, capsules, suppositories, or dispersible granules. A solid carriercan be one or more substances that function as a diluting agent, flavoradditive, solvent, lubricant, suspension agent, binder, preservative,tablet-disintegrating substance or encapsulating material.

In powdered form, the carrier is a finely pulverized solid includinglactose, hydroxypropylmethylcellulose and PVP, mixed with an appropriateamount of finely pulverized lectin preparation.

Appropriate carriers for powder and tablet forms include magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,stiffeners, gelatins, tragacanth, methylcellulose, and sodiumcarboxymethylcellulose. The term “composition” is meant to includedosage-forms where the active ingredients are enclosed in anencapsulating material whether or not associated with a carrier,including capsules or lozenges.

Suppositories are produced by melting a low-melting point wax anddistributing the lectin therein. The melted, homogeneous mixture is thenpoured into forms and allowed to cool.

Compositions appropriate for vaginal administration can be presented aspresses, tampons, creams, gels, pastes, foams or sprays that include, inaddition to the active ingredient, suitable carriers known in the art.

Compositions in liquid form include solutions, suspensions, andemulsions, for example aqueous or propylene glycol solutions, togetherwith coloring agents, flavor additives, stabilizing agents or dilutingagents as appropriate. Also included are compositions in solid form thatare meant to be converted to liquid form shortly prior to consumption.These forms may include, in addition to the active ingredients,artificial colors, flavors, stabilizers, buffers, natural or artificialsweeteners, dispersing agents, thickeners, dissolving agents and thelike.

For topical administration to the epidermis, the lectin composition canbe presented in the form of salves, creams, gels, skin washes ortransdermal plasters. Salves and creams can be formulated with anaqueous or oil base, with the addition of suitable thickeners and/orgels. Skin washes can be prepared with an aqueous or oil base and maycontain one or more emulsifying agents, stabilizers, dispersing agents,thickeners or fragrances.

Compositions suitable for topical administration in the mouth includelozenges that include active ingredients in an inert, flavored base,such as sucrose and Arabica gum, as well as mouth washes containing theactive ingredients in a liquid carrier.

Solutions or mixtures may be administered directly to the nasal cavityusing conventional means, such as drops or sprays. The composition maybe produced in individual or multi-dose forms. Multi-dose forms wouldinclude a dropper, pipette or atomizer that delivers a predeterminedvolume of the composition.

Administration to the respiratory tract can be achieved by the use of anaerosol preparation in which the active ingredient is placed in apressurized container together with a suitable delivery agent, such asCFC, trichlorofluormethane, dichlorofluormethane, carbon dioxide orother suitable gas. The dosage may be controlled by an appropriatevalve-system.

The pharmaceutical composition is preferably provided in individualdosage units that contain a suitable amount of the active ingredient.The individual doses may be provided in a package, or as a kit thatincludes a measuring device, e.g., a device for measuring oral orinjectable dosages (i.e., a measuring cup, needle, or syringe). The kitcan also include, other materials such buffers, diluents, filters, andpackage inserts with instructions for use. A label may be present on theon the kit to indicate that the composition is used for a specifictherapy, and may also indicate directions for use, such as thosedescribed above.

The compositions and methods of the present invention may also be usefulin reducing the side effects of traditional chemo-radio therapies,wherein lectin is administered in conjunction with the chemo-radiotherapy thereby reducing the amount of toxic dosage needed to killcells.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Materials and Methods

Cell Culture.

Nontransformed and Src transformed wild-type mouse embryonic fibroblasts(WT), homozygous null Cx43 knockout brain cells (Cx43Ko), and homozygousnull Cas knockout cells (CasKo) are known in the art (Goldberg, et al.(2003) J. Biol. Chem. 278:46533-46540; Shen, et al. (2006) Cancer Res.66:1543-1552; Alexander, et al. (2004) Cancer Res. 64:1347-1358;Patwardhan, et al. (2006) J. Biol. Chem. 281:20689-20697). LA25 cellsharboring a temperature sensitive v-Src construct were grown asdescribed (Crow, et al. (1992) Oncogne 7:999-1003). B16F10 melanomacells were obtained from ATCC, and melan-a melanocytes were as describedin the art (Estler, et al. (2008) BMC Genomics 9:478). cDNA encodingmurine Pdpn was released from pCMV-Sports-pdpn (Open Biosystems#MMM1013-7513215) with EcoRI and Xba1 and inserted into thecomplementary sites of pEF4 to create pEF4Pdpn. B16 cells weretransfected with pEF4Pdpn, empty pEF4 vector, 200 nM siRNA directedagainst mouse Pdpn, or 200 nM control siRNA (Dharmacon, Chicago, Ill.)with LIPOFECTAMINE 2000 (Li, et al. (2008) Cancer Sci. 99:1326-1333;Shen, et al. (2007) J. Biol. Chem. 282:18914-18921). Cells transfectedwith pEF4 expression vectors were selected with zeocin. Clones were nottaken from any cell lines, thus minimizing potential effects of clonalvariation. In some experiments, cells were treated with MAA (SigmaL8025, Sentrimed MASL) or puromycin (Sigma P8833).

Layered Culture System.

A Layered Culture System was used to allow separated populations oftransformed and nontransformed cells to form intercellular junctionswith each other as described (Alexander, et al. (2004) supra; Goldberg,et al. (2002) J. Biol. Chem. 277:36725-26730). Briefly, ten thousand Srctransformed cells were plated on porous membranes (Costar) containing300 thousand nontransformed cells on the other side. Transformed cellsand nontransformed cells were able to form intercellular junctionsthrough the pores in the membrane; however, the membrane pore size (3pm) is small enough to prevent cells (about 20 pm in diameter) fromactually migrating to the other side of the membrane (Shen, et al.(2006) supra; Alexander, et al. (2004) supra; Goldberg, et al.(2002)supra). Transformed cells were also plated alone, directly above300 thousand nontransformed cells, or 1 mm above 300 thousandnontransformed cells as controls. Nontransformed cells were also platedon these membranes as controls. Cells were harvested and analyzed 24hours after plating.

Expression Microarrays.

Gene expression in nontransformed, Src transformed, and contactnormalized Cx43Ko cells was examined by microarray analysis with 430 2.0Mouse Expression Array gene chips (Affymetrix) using conventionalmethods (Shen, et al. (2006) supra). These arrays contain approximately45,000 probe sets which represent over 30,000 genes. Affected probe setsdisplayed a difference of at least 4-fold between transformed andcontrol cells, or at least a 2-fold change with p<0.05 by t-test withn=3. Genes that were increased by Contact Normalization were alsodecreased by Src, but not affected by diffusible factors fromnontransformed cells or contact with other transformed cells.Conversely, genes that were decreased by Contact Normalization wereincreased by Src, but not affected by diffusible factors fromnontransformed cells or contact with other transformed cells. Allcomparisons were done with cells from parallel cultures to control forvariability in reagents or experimental conditions. Expression analysiswas performed with Vector Xpression software 4.0 (Invitrogen).

RT-PCR.

RNA was purified with Tri-reagent (Sigma, St. Louis, Mo.). cDNA wassynthesized from 1 μg of RNA by with PROTOSCRIPT First Strand cDNASynthesis Kit (NEB, Ipswich, Mass.). PCR was performed with 1 μl of cDNAwith forward and reverse primers specific for GAPDH (5′-TGC ATCCTG CACCAC CAA CT-3′, SEQ ID NO:1; and 5′-TGC CTG CTT CAC CAC CTT C-3′, SEQ IDNO:2), hygromycin phosphotransferase (5′-CAT GGC GTG ATT TCA TAT GCGCGA-3′, SEQ ID NO:3; and 5′-TCC AGA AGA AGA TGT TGG CGA CCT-3′, SEQ IDNO:4), puromycin acetyltransferase (5′-ACC GAG CTG CAA GAA CTC TTCCTC-3′, SEQ ID NO:5; and 5′-AGG AGG CCT TCC ATC TGT TGC T-3′, SEQ IDNO:6), Pdpn (5′-ACC AAC ACA GAC GAC CAA GAC ACT-3′, SEQ ID NO:7; and5′-AAG CAT CCA CTG TGC CTT CAG TTC-3′, SEQ ID NO:8), Fhll (5′-GAG AAGTTC GAC TGT CAC TAC TGC-3′, SEQ ID NO:9; and 5′-CTG ATC CTG GTA AGT GATTCC TCC-3′, SEQ ID NO:10), 4930408021Rik (5′-TGT TCT CAG AGC CCA GCA TCACTT-3′, SEQ ID NO:11; and 5′-ACA TCC TCT CAG CTG OTT CCT TCA-3′, SEQ IDNO:12), 1700012B09Rik (5′-CTG TGA ACC GCA TAA GAG AAT CAA GGA GG-3′, SEQID NO:13; and 5′-TGC CTC GAG TAG TAC TTG GCT TGT-3′, SEQ ID NO:14),Tmem163 (5′-ATA GAG TCT GTC ATC ATG GGC TGG-3′, SEQ ID NO:15; and 5′-ACAGGC TTC CTG TCA AGC AGA GA-3′, SEQ ID NO:16), Loc677224 (5′-AAC ATC CCAGAG CCT TTG ACT CCT-3′, SEQ ID NO:17; and 5′-CAA AGC TGC CAT AGC TCT ATTCGG-3′, SEQ ID NO:18), 1810008K04Rik (5′-AAG CCA GGA CTC TCA CAT GCAACT-3′, SEQ ID NO:19; and 5′-AGC TTT GCA GAT GGA ACG GAA CAC-3′, SEQ IDNO:20), and D4bwg0951e (5′-TGG ATG GCA TCT CAG TAG GGA GCT A-3′, SEQ IDNO:21; and 5′-TTG CAC ACC AGT CCC ATG CAA A-3′, SEQ ID NO:22). GAPDH wasamplified at 95° C. for 5 minutes, 95° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 30 seconds for 30 cycles, followed by 72° C. for5 minutes. Hygromycin phosphotransferase was amplified at 95° C. for 5minutes, 95° C. for 30 seconds, and 72° C. for 1 minute for 30 cycles,followed by 72° C. for 5 minutes. The other genes were amplified at 95°C. for 5 minutes, 95° C. for 30 seconds, 60° C. for 30 seconds, and 72°C. for 30 seconds for 30 cycles, followed by 72° C. for 5 minutes.

Western Blot Analysis.

Western blot analysis was performed according to conventional methods(Shen, et al. (2006) supra; Alexander, et al. (2004)Cancer Res.64:1347-58). Protein (10 lag/lane) was resolved by SDS-PAGE, transferredto IMMOBILON-P membranes (Millipore), and incubated with antiseraspecific for Pdpn (Santa Cruz Biotechnology), Cas (Santa CruzBiotechnology), active Src kinase (Cell Signaling Technology), PARP,(Cell Signaling Technology), v-Src (Upstate Biotechnology), or β-actin(Sigma). Primary antiserum was recognized by appropriate secondaryantiserum conjugated to horseradish peroxidase and detected usingEnhanced Chemiluminescence (Millipore). After blotting, membranes werestained with India ink to verify equal loading and transfer.

Immunofluorescence Microscopy.

Cells (300,000 per dish) were cultured on 35-mm poly-D-lysine-coatedglass-bottomed culture dishes (MatTek) for 24 hours. For someexperiments, lectin from Maackia amurensis (Sigma L8025) was conjugatedto HILYTE FLUOR TR (Anaspec) and incubated with cells for 15 minutes at37° C. Cells were fixed with 2% paraformaldehyde, permeabilized with0.2% TRITON X-100 in PBS (phosphate-buffered saline) for 10 minutes,washed thrice with 0.1% TWEEN 20 in PBS followed by 1% bovine serumalbumin (BSA) in PBS for 30 minutes, incubated with Pdpn antiserum(1:100) overnight at 4° C., washed, and then labeled with goatanti-syrian hamster IgG conjugated to ALEXA FLUOR 488 (MolecularProbes). Cells were visualized on ZEISS AXIOVERT or PASCAL microscopesystems (Li, et al. (2008) supra; Shen, et al. (2007) supra).

Cell Growth, Migration, and Toxicity Assays.

Cell monolayers were scratched and incubated with MAA for 24 hours toassess cell migration by wound healing assays, which were quantitated asthe number of cells that entered an area of the wound indicated asdescribed (Shen, et al. (2006) supra; Shen, et al. (2010) supra). Alamarblue (INVITROGEN DAL1025) was added to cells 24 hours after MAAtreatment, and incubated for an additional 8 hours to assay viability,which was detected by fluorescence measurements (ex/em 570/600 nm) asdirected by the manufacturer (INVITROGEN) in a TECAN GENIOS microplatefluorescence spectrophotometer. Cells were also stained with 0.2% trypanblue and counted with a hemocytometer to evaluate cytotoxicity. Tomeasure cell invasion, 600,000 cells were plated in 6-well clusterplates on cell culture inserts with an 8 micron pore size(Transwell-Clear, Costar) and grown for 24 hours. Cells were thenreleased separately from the top of the membrane and the bottom of themembrane. Invasion was then quantitated as the percent of cells found onthe underside of the membrane as described (Shen, et al. (2010) supra).All experiments were performed on parallel cultures to control forvariations in cell culture conditions.

Affinity Precipitation.

Cells were lysed in lysis buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl,0.5% TRITON X-100, 1 mM PMSF) on ice for 30 minutes, clarified bycentrifugation, diluted to 1 mg/ml in PBS supplemented with 1 mM PMSFand 10 mM MgCl2, and incubated with lectin from M. amurensis (Sigma)conjugated to agarose beads or empty beads as controls (ThermoScientific), on ice for 3 hours. Beads were then washed four times withPBS, and eluted in of SDS-PAGE sample buffer at 95° C. for 5 minutes.Eluted protein was examined along with nonprecipitated cell lysates bywestern blot analysis.

In Vivo Tumorigenesis.

Mice (C57BL/6) were fed with a 200 mg food pellet containing 0 or 25mg/kg MAA once weekly, starting the day before subcutaneous inoculationwith 100,000 B16 cells in 0.1 ml PBS. Tumor volumes were measuredblindly by a trained physician with a single caliper daily. Mice weresacrificed 18 days after inoculation and dissected. Tumors were fixed informalin, paraffin embedded, sectioned (5 microns), and processed forhematoxylin/eosin staining or immunohistochemistry to detect PDPNexpression with 8.1.1 monoclonal antibody as described (Shen, et al.(2006) supra; Shen, et al. (2010) supra). Samples were analyzed on aZEISS AXIOVERT microscope with AXIOVISION software as described (Shen,et al. (2006) supra; Shen, et al. (2010) supra).

Ex Vivo Effects of Dietary MAA on Cell Migration.

Mice (C57BL/6) were fed with a 200 mg food pellet containing 0, 100, or200 mg/kg MAA 1 hour before blood was taken from subclavian vein. Bloodwas allowed to coagulate 1 hour at room temperature and clarified bycentrifugation for 10 minutes at 20,000 g to obtain serum. This serumwas added to cell culture medium (DMEM+10% FBS) to a final concentrationof 20%. B16 cell monolayers were scratched, washed with DMEM, and thenincubated with these media, or control medium not fortified with mouseserum, for 24 hours to assess their effects on cell migration by woundhealing assays, which were quantitated as the number of cells thatentered an area of the wound (Shen, et al. (2006) supra; Shen, et al.(2010) supra).

Example 2 Screening Assay for Genes Associated with ContactNormalization

Tumor cells must overcome contact normalization by adjacentnontransformed cells in their microenvironment to become malignant ormetastatic (Rubin (2007) Adv. Cancer Res. 98:117-147). Using a layeredculture system, identify genes involved in this process (Shen, et al.(2006) Cancer Res. 66:1543-1552; Alexander, et al. (2004) Cancer Res.64:1347-1358) were identified. As shown in FIG. 1, Src transformed cellswere incubated on a porous membrane directly over nontransformed cells.The transformed cells and nontransformed cells were able to formintercellular junctions through the pores in the membrane while beingretained as separate populations. Thus, although intercellular junctionsformed between the transformed and nontransformed cells, the layers wereretained as distinct populations that were separately harvested. Ascontrols, transformed cells were also incubated alone, or 1 mm overnontransformed cells to enable communication by diffusible factors butnot direct contact. As has been previously reported, cellular materialis not transferred between harvested cell layers (Shen, et al. (2006)supra; Alexander, et al. (2004) supra; Goldberg, et al. (2002) J. Biol.Chem. 277:36725-36730). This was verified by the retention of the Srckinase and mRNA encoding puromycin acetyltransferase in the transformedcells, and mRNA encoding hygromycin phosphotransferase in nontransformedcells.

Using AFFYMETRIX microarrays, mice genes involved in ContactNormalization were identified. About 45,000 probe sets were examined,representing over 39,000 transcripts. The expression of genesrepresented by 10928 probe sets, or up to about 25% of thetranscriptome, was affected 2-fold or more by Src. The expression ofgenes represented by up to 5560 probe sets was higher in Src transformedcells than nontransformed cells, while the expression of genesrepresented by up to 5368 probe sets was lower. However, only 39 geneswere found to be significantly affected by Contact Normalization of Srctransformed cells (Table 2). The mRNA levels of several of these genes,including Pdpn, Tmem163, Fhll, and 5 ESTs were verified by RT-PCR. Theexpression of these genes was clearly affected by Src and reversed bycontact with nontransformed cells.

TABLE 2 Nucleotide Fold Change Gene Symbol Accession No. Src^(a) CN^(b)Genes Increased by Src and Decreased During Contact Normalization1700012B09Rik^(c) NM_029306 115 −3.4 Pdpn^(c) NM_010329  93 −4.7Tmem163^(c) NM_028135  58 −5.5 Smoc2 NM_022315  47 −6.6 Pstpip2NM_013831  41 −4.5 Mal NM_010762  30 −5.6 Lepr NM_146146  28 −4.7 Elavl2NM_207685  26 −9.9 Igsf11 NM_170599  26 −5.0 Mafb NM_010658  14 −6.7LOC677224^(c) XR 034093.  13 −3.5 4930408O21Rik^(c) NM_001040136    8.8−4.8 Elmod1 NM_177769    8.3 −5.0 Atpl3a4 NM_172613    7.8 −4.7 KdrNM_010612    6.6 −3.7 C9 NM_013485    5.4 −4.9 Genes Decreased by Srcand Increased During Contact Normalization Fgf7 NM_008008 −130  46  D10Bwg1379e NM_001033258 −95 13   Soxll NM_009234 −93 16   Cc120NM_016960 −91 12   Vcam1^(d) NM_011693  −78* 22*   −56* 16*   −55* 12* Sema3e NM_011348 −71  8.3 Sdpr^(d) NM_138741 −47 13*  −39 11*  Epha4NM_007936 −44  8.3 Ntnl NM_008744 −42 17   Cabcl NM_023341 −32  5.0Spryl NM_011896 −26  4.5 Lox11 NM_010729 −25  9.9 1810008K04Rik^(c)NM_001081050 −19  9.0 Pdgfrb NM_008809 −18  3.9 D4Bwg0951e^(c) NM_026821−17  4.2 Fhl1^(c,d) NM_001077361 −16 11   Gpr126 NM_001002268  −16*  5.7*  −13*   5.7* Egfr NM_207655 −16  5.2 Itih2 NM_010582 −12  7.3Epb4.113 NM_013813 −12  5.6 Illrll NM_001025602   −9.7  8.3 Wnt5aNM_009524   −9.1  4.6 Tmem56 NM_178936   −8.2  4.4 ^(a)Expression in Srctransformed cells divided by expression in nontransformed cells.^(b)Expression in transformed cells divided by expression in transformedcells undergoing Contact Normalization. ^(c)Effects confirmed by RT-PCR.^(d)Genes previously shown to be induced by Contact Normalization.*Represent results with different probe sets.

As shown in Table 2, the expression of 23 genes was suppressed by Srcand increased by contact normalization. Some of these genes encodeproteins that mediate events including growth factor signaling (Fgf7,Epha4, Spryl, Pdgfrb, Gpr126, Egfr, Wnt5a, and Illr11) and transcription(Soxll and Fhll). Many of these genes have been implicated in theregulation of tumor cell growth and migration. For example, recentexperiments indicate that Epb4.113 (erythrocyte protein band 4.1-like 3)(Cavanna, et al. (2007) J. Cell Sci. 120:606-616) and Itih2(inter-alpha-trypsin inhibitor heavy chain) (Werbowetski-Ogilvie, et al.(2006) Cancer Res. 66:1464-1472) can suppress tumor cell invasion andmetastasis. Induction of three of these genes, Vcaml, Sdpr, and Fhll isassociated with contact normalization (Alexander, et al. (2004) supra).Interestingly, these genes are candidate biomarkers that are suppressedin malignant tumor cells (Li, et al. (2008) Cancer Sci. 99:1326-1333).For example, Src utilizes Cas to suppress Fhll expression in order topromote tumor cell migration (Shen, et al. (2006) supra).

The expression of 16 genes was increased by Src and suppressed bycontact normalization. These genes are of great interest since they maybe required for malignant growth and metastasis. These genes encodeproteins that mediate events including cation (Smoc2, Atp13a4) andglutamate (Tmem163) transport, neuronal development (Mal), energyhomeostasis (Lepr), mRNA transcription (Mafb) and stabilization(Elav12), cell adhesion (Igsf11), growth factor signaling (Kdr), andimmunological activity (C9). Some of these gene products may act asbiomarkers and targets for chemotherapy. For example, inhibitors havebeen generated to target kinase insert domain receptor (Kdr) in order toprevent VEGF signaling and suppress angiogenesis required for malignanttumor growth (Schenone, et al. (2007) Curr. Med. Chem. 14:2495-2516).Immunological reagents have also been targeted to immunoglobulinsuperfamily 11 (IGSF11) (Watanabe, et al. (2005) Cancer Sci. 96:498-506)and podoplanin (Pdpn) (Kato, et al. (2008) Cancer Sci. 99:54-61) inorder to prevent tumor cell invasion and metastasis.

Since the gap junction protein Cx43 can affect transformed cellmigration (Xu, et al. (2006) Development 133:3629-3639; Shao, et al.(2005) Cancer Res. 65:2705-2711), the effects of Src on Pdpn expressionwere analyzed in Cx43Ko (knock out) and wild-type cells. While more Pdpnwas found in wild-type cells than Cx43Ko cells, western blot analysisrevealed that Src augmented Pdpn protein expression in both cell types.These findings were confirmed by immunofluorescence microscopy.

These data indicate that Pdpn acts downstream of Src and Cas along thepathway leading to nonanchored tumor cell growth and migration. Thisstudy presents an example of how genes that are induced by Src andsuppressed by Contact Normalization can be utilized as biomarkers andpromising targets for chemotherapy. These genes also find application inassessing the role of the proteins encoded thereby in malignant growthpotential.

Example 3 Src Utilizes Cas to Induce Pdpn Expression

Pdpn expression was notably affected by Src and Contact Normalization.This was intriguing since Pdpn can promote tumor cell migration leadingto malignant and metastatic growth (Wicki, et al. (2007) Br. J. Cancer96:1-5; Kato, et al. (2008) supra; Nakazawa, et al. (2008) Blood112(5):1730-9). It has been found that Src utilizes Cas to suppress Fhllexpression in order to promote nonanchored cell growth and migration ofSrc transformed cells (Shen, et al. (2006) supra). Therefore, theeffects of Src on Pdpn expression were examined in cells with andwithout Cas. This analysis indicated that more Pdpn was found in Srctransformed cells CasKo cells transfected with Cas than controltransfectants. These data indicate that Src utilizes Cas to augment Pdpnexpression and promote tumor cell migration.

The effects of Pdpn on the motility of Src transformed cells wereexamined by transfection with siRNA. This analysis indicated that Pdpnprotein expression was decreased in Src transfected cells transfectedwith siRNA directed against Pdpn. Wound healing assays showed that thisdecrease in Pdpn expression reduced the migration of Src transformedcells by over 60%. These data indicate that Src induced Pdpn expressionto promote cell migration.

The effects of Pdpn on the motility of nontransformed cells were alsoexamined by transfection with cDNA. Western blot analysis demonstratedthat nontransformed cells, transfected with cDNA encoding Pdpn,expressed Pdpn protein levels comparable to Src transformed cells.Immunofluorescent microscopy found Pdpn localized to the plasma membranewhere it may act as a signaling receptor. Pdpn expression did notsignificantly affect the growth or anchorage dependence ofnontransformed cells. However, Pdpn expression did augment the abilityof nontransformed cells to migrate by over 5 fold, achieving levelscomparable to Src transformed cells. Thus, Pdpn did not requiretransforming Src activity to promote cell migration. Nonetheless, sincePdpn did not augment nonanchored growth, it was not sufficient toincrease colonization of nontransformed cells.

In addition to Src transformed mouse cells, Pdpn expression levels werealso examined along with the migration of human mammary carcinoma cells.This analysis indicated that levels of Pdpn expression correlated wellwith the migration of MCF7, MDA-MB-231, BT-549 cells shown in FIG. 2.These data are consistent with Pdpn augmenting the migration ofepithelial tumor cells.

Example 4 Model Systems for Analyzing Biomarkers and ChemotherapeuticTargets

Cell systems of use in identifying biomarkers and chemotherapeutictargets to specifically detect and neutralize malignant and metastaticcancer cells that are not controlled by Contact Normalization are listedin Table 3. Using such systems it has been found that Src induces theexpression of Pdpn and promotes anchorage indendence and motilityrequired for tumor cell invasion metastasis. It has also beendemonstrated that some plant lectins can bind to podoplanin and inhibitnonanchored tumor cell growth and motility at nontoxic concentrations.Therefore, natural products and compounds derived from natural productscan be used in the prevention of malignant progression without harmingother cells of the body.

TABLE 3 Anch. Migra- Cells Src Cas Pdpn Independ. tion CasKO (homozygousnull Cas knockout cells) CasKOHP − − − − − CasKOHP-Pdpn (−) (−) (+) (+)(+) CasKOHP-Ef4 (−) (−) (−) (−) (−) CasWt − + − − − CasKOSrc + − − − −CasWtSrc + + + + + CasWtSrc-siCas (+) (−) (−) (−) (−) CasWtSrc-siPdpn(+) (+) (−) (−) (−) CasWtSrc-siCntl (+) (+) (+) (+) (+) MEF (mouseembryonic fibroblasts) Mef − + − − − Mef-Pdpn (−) (+) (+) (+) (+)Mef-Ef4 (−) (+) (−) (−) (−) MefSrc + + + + + MefSrc-siCas (+) (−) (−)(−) (−) MefSrc-siPdpn (+) (+) (−) (−) (−) MefSrc-siCntl (+) (+) (+) (+)(+) LA25 (temperature sensitive Src transformants) LA25-34 C + + + + +LA25-siCas-34 C + − (−) (−) (−) LA25-siPdpn-34 C (+) (+) (−) (−) (−)LA25-siCntl′-34 C + + (+) (+) (+) LA25-39 C − + − − − LA25Ef4-39 C (−)(+) (−) (−) (−) LA25Pdpn-39 C (−) (+) (+) (+) (+) Anticipated resultsare shown in parentheses.

Example 5 Targeting Podoplanin with a Plant Lectin to Inhibit Tumor CellGrowth and Migration

PDPN is a transmembrane mucin glycoprotein receptor that augments tumorcell migration. PDPN expression can be induced by tumor promotersincluding TPA, oncogenic Ras, and Src (Nose, et al. (1990) Cell GrowthDiffer. 1:511-518; Gandarillas, et al. (1997) Mol. Carcinog. 20:10-18;Shen, et al. (2010) J. Biol. Chem. 285:9649-9656). PDPN regulates theactivities of effectors including ezrin, Rho, and Cdc42 to mediatefilopodia formation and promote tumor cell metastasis (Wicki &Christofori (2007) Br. J. Cancer 96:1-5; Martin-Villar, et al. (2009)Int. J. Biochem. Cell. Biol. 41:1421-29; Navarro, et al. (2010) Am. J.Physiol. Lung Cell. Mol. Physiol. 300(1):L32-42).

PDPN is found at the invasive front of many tumors, which is consistentwith its role in promoting malignant invasion (Martin-Villar, et al.(2005) Int. J. Cancer 113:899-910; Wicki, et al. (2006) Cancer Cell9:261-272; Wicki & Christofori (2007) supra). For example, PDPNexpression is strongly induced in about 40% of breast cancers (Wicki, etal. (2006) supra; Kono, et al. (2007) Int. J. Oncol. 31:501-508), 50% oforal cancers (Martin-Villar, et al. (2005) supra; Yuan, et al. (2006)Cancer 107:563-9; Kawaguchi, et al. (2008)J. Clin. Oncol. 26:354-360),and 80% of skin cancers (Schacht, et al. (2005)Am. J. Pathol.166:913-921; Liang, et al. (2007) Am. J. Surg. Pathol. 31:3014-310). Thebulk of the PDPN protein, about 150 amino acids, lies outside of thecell and could serve as an ideal target to combat cancer invasion andmetastasis (Wicki & Christofori (2007) supra; Kunita, et al. (2007) Am.J. Pathol. 170:1337-1347).

Antibodies against PDPN can inhibit lung metastasis of transformed cellsthat express PDPN (Nakazawa, et al. (2008) Blood 112(5):1730-9; Kato, etal. (2008) Cancer Sci. 99:54-61). However, alternatives to antibodiesare needed (Johnson & Brown (2010) Semin. Oncol. 37:345-58). As analternative, lectins are resistant to gastrointestinal proteolysis andcan be taken orally to affect a wide range of diseases including cancer.Indeed, dietary lectins can block the action of endogenouspro-metastatic lectins (such as galectins or selectins) to inhibit tumorcell growth. However, most anti-cancer lectins that have been examinedthus far target receptors that have not been identified (Pryme, et al.(2006) Histol. Histopathol. 21:285-99; Hasan, et al. (2007) Cancer Lett.253:25-33; Pusztai, et al. (2008) Front. Biosci 13:1130-40; Liu, et al.(2010) Cancer Lett. 287:1-12).

The extracellular domain of PDPN is highly 0-glycosylated with sialicacid, α2,3 linked to galactose and has been shown to associate withlectin from the legume tree M. Amurensis (MAA) and promote cellmigration (Scholl, et al. (1999) J. Cell Sci. 112 (Pt24):4601-13; Wicki& Christofori (2007) supra). It has now been shown that a lectin withaffinity for O-linked carbohydrate chains containing sialic acid bindsto PDPN to inhibit the growth and migration of melanoma cells atnanomolar concentrations. This approach demonstrates how lectins may beused as dietary agents that target specific receptors to combatmalignant cell growth.

The analysis described here was carried out with Src transformed cells,which displayed strong PDPN expression and motility (Shen, et al. (2010)J. Biol. Chem. 285:9649-56). The results of this analysis indicated thatMAA associated with PDPN on the membrane of Src transformed cells. Notethat MAA did not target the membrane of cells that did not express PDPN,which, along with functional studies described herein, attests to itspreferential targeting. In addition, MAA associated with PDPN in lysatesfrom transformed cells during affinity precipitation experiments.Moreover, using human melanoma cells that express Pdpn, it was foundthat the growth of LOX IMVI and SK-MEL-5 cells, which express relativelyhigh levels of Pdpn, was inhibited by greater than 85% with 325 nM orless MAA, whereas growth of UACC-257 cells, which express relativelylower levels of Pdpn, was inhibited by less than 20%.

As has been reported (Shen, et al. (2010) supra), SRC transformed cellsexpressed more PDPN and migrated more than nontransformed controls.Therefore, it was determined whether binding of MAA to PDPN wouldinhibit transformed cell migration. The MAA used in this analysis hadthe unique sequence: SDELSFTINN FVPNEADLLF QGEASVSSTG VLQLTRVENGQPQQYSVGRA LYAAPVRIWD NTTGSVASFS TSFTFVVKAP NPTITSDGLA FFLAPPDSQIPSGRVSKYLG LFNNSNSDSSNQIVAVEFDT YFGHSYDPWD PNYRHIGIDV NGIESIKTVQWDWINGGVAF ATITYLAPNK TLIASLVYPS NQTSFIVAAS VDLKEILPEW VRVGFSAATGYPTQVETHDV LSWSFTSTLE ANCDAATEN (SEQ ID NO:23).

This analysis indicated that MAA significantly inhibited transformedcell migration, with 385 nM, 770 nM, and 1540 nM inhibiting cellmigration by over 25%, 50%, and 75%, respectively (p<0.0002 by ANOVA).In addition to inhibiting cell migration, MAA also inhibited the growthof Src transformed cells in a dose-dependent fashion (p<0.0001 ANOVA).In contrast, MAA did not inhibit the growth of nontransformed cells inan equally dose-dependent fashion (p=0.28 by ANOVA).

To verify the functional relevance of MAA targeting PDPN on cellmigration, the effect of MAA (385, 770 and 1540 nM) on nontransformedcells transfected with PDPN or empty parental vector was investigated.Since PDPN expression is sufficient to promote cell migration (Scholl,et al. (1999) supra; Wicki & Christofori (2007) supra; Shen, et al.(2010) supra), nontransformed cells transfected with PDPN migratedseveral fold more than control transfectants. In addition, MAA reducedthe migration of these nontransformed PDPN transfected cells in adose-dependent fashion (p<0.0001 by ANOVA). For example, 385 nM MAAdecreased the migration of PDPN transfectants by over 40%.

In addition to inhibiting cell migration, MAA was also toxic to PDPNexpressing cells in a dose-dependent fashion (p<0.0001 ANOVA). Incontrast, MAA did not inhibit the viability of empty vectortransfectants in an equally dose-dependent fashion. For example, 1540 nMMAA decreased Trypan blue exclusion of PDPN transfectants by over 70%,but control transfectants by only about 30%.

Studies indicate that PDPN expression is strongly induced in about 80%of skin cancers (Schacht, et al. (2005) supra; Liang, et al. (2007)supra). Consistent with its role in tumor cell invasion and metastasis,malignant B16 melanoma cells expressed higher levels of PDPN andmigrated significantly better than nontransformed Melan-a melanocytes(more than 25 cells/field vs. 2.5 cells/field). MAA effectivelysuppressed melanoma cell migration at concentrations of 308 nM or less.In addition, MAA inhibited melanoma cell growth in a dose-responsivemanner (p<0.0001 by ANOVA). Moreover, MAA was significantly more toxicto B16 melanoma cells than Melan-a cells (p=0.0005 by ANOVA). Aninvasion chamber was used to further investigate the effects of MAA onmelanoma cell growth and migration. While 385 nM MAA was notsignificantly toxic to B16 melanoma cells (p>0.2 compared to controls),invasion through 8 micron pores was decreased by over 40-fold.

To verify the effects of PDPN and MAA on melanoma cell growth andmigration, siRNA was employed. This analysis showed that PDPN siRNAeffectively decreased B16 Pdpn expression levels and cell migration.This decreased PDPN expression resulted in a 25% decrease to MAAtoxicity. These data indicated that while PDPN may not be the only MAAtarget expressed by these melanoma cells, it is a functionally relevantreceptor that can be targeted to prevent melanoma cell growth andmigration.

Since lectins are resistant to gastrointestinal proteolysis (Pryme, etal. (2002) supra; Pusztai, et al. (2008) supra), the effects of dietaryMAA on tumor cell growth in vivo were examined. Oral administration of25 mg/kg of MAA once a week inhibited the subcutaneous growth ofmelanoma cells in mice by approximately 50% (p<0.05 by ANOVA). Moreover,no adverse effects on mouse health or physiology were observed over thecourse of these experiments based on animal behavior, weight, and organanalysis after dissection.

PDPN expression was evident in melanoma cells of tumors in vivo and wasmore intense in tumor cells from control animals than animals treatedwith MAA. In addition, tumor cells from MAA-treated animals appearedmore epithelial in nature, with a relatively flattened morphology andmore restricted PDPN staining than tumors from untreated animals.Interestingly, blood filled vascular spaces lined by tumor cells weremuch smaller and less numerous in tumors from MAA-treated mice than fromcontrol animals. Specifically, tumor vascularization was quantified asthe percent of each field (0.8 mm²) occupied by blood vessels it wasobserved that samples from MAA-treated mice had fewer than then 5% bloodvessels in each field compared to 36% in control animals. Thus, likesome other anticancer agents, MAA appeared to inhibit blood tumorvascularization, which may contribute to its ability to inhibit tumorgrowth (Rybak, et al. (2003) Cancer Res. 63:2812-2819). Theseexperiments have been repeated with similar results.

Serum was taken from mice fed MAA and then examined for its effects onmelanoma cell migration in vitro to verify that this dietary lectin cansurvive gastrointestinal proteolysis to remain biologically active inthe circulatory system. This analysis indicated that serum from mice fed100 or 200 mg/kg MAA inhibited melanoma cell migration by about 30% orover 80% compared to mice fed no MAA, respectively (p<0.0001 by ANOVA).These data clearly indicate that dietary administration of MAA canresult in biologically relevant levels of circulating product that aresufficient to inhibit melanoma cell migration.

To demonstrate the broad applicability of MAA against different cancers,the effects of MAA on human mammary carcinoma cells was also examined.This analysis indicated that MAA was more effective on BT-549 andMDA-MB-231 cells, which expressed high levels of Pdpn, than MCF-7 cells,which expressed lower levels. The interaction between cell type and MAAexposure accounted for 18% of the variation of migration between samples(FIG. 2A) and 10% of the variation of toxicity between samples (FIG. 2B)(p<0.0001 by two-way ANOVA).

The lectin jacalin, derived from the jackfruit tree Artocarpusintegrifolia, was used as a control in some of these studies because itpreferentially binds to glycoconjugates containing galactosyl(31,3)N-acetylgalactosamine (Lehmann, et al. (2006) Cell Mot. Life Sci.63:1331-1354). In contrast to MAA, jacalin does not reduce cellmigration at nontoxic concentrations and appeared to augment cellmigration at concentrations of up to 400 nM. Thus, suppression of tumorcell migration appeared to be lectin-specific and consistent withbinding to extracellular moieties presented by Pdpn.

To further demonstrate the use of lectin in the treatment of cancer GIsovalues were determined using a panel of more than 50 cancer cell lines.GI₅₀ values represent the concentration of the anticancer agent thatinhibits the growth of cancer cells by 50% (in other words, after givingthe agent, there is a 50% reduction in cancer cell proliferation). Thisanalysis indicated that MAA inhibits the growth of cancer cellsincluding leukemia, non-small cell lung cancer, colon cancer, CNScancer, melanoma, ovarian cancer, renal cancer, prostate cancer andbreast cancer (Table 4).

TABLE 4 Cancer Cell Line GI₅₀ Leukemia CCRF-CEM 6.56E−8 HL-60(TB)6.67E−8 K-562 >7.68E−7  MOLT-4 5.71E−8 RPMI-8226 >7.68E−7  SR 8.93E−8non-small cell A549/ATCC 3.87E−7 lung cancer EKVX 3.43E−7 HOP-62 3.18E−7HOP92 1.30E−7 NCI-H226 >7.68E−7  NCI-H23 >7.68E−7  NCI-H322M >7.68E−7 NCI-H460 3.80E−7 NCI-522 >7.68E−7  colon cancer COLO 205 >7.68E−7 HCC-2998 >7.68E−7  HCT-116 3.46E−7 HCT-15 9.47E−8 KM12 6.39E−7SW-620 >7.68E−7  CNS cancer SF-268 2.32E−7 SF-295 4.92E−7 SF-539 1.90E−7SNB-19 4.83E−7 SNB-75 2.05E−7 U251 1.67E−7 Melanoma LOX IMVI 1.01E−7MALME-3M >7.68E−7  M14 3.46E−7 MDA-MB-435 3.85E−7 SK-MEL-2 >7.68E−7 SK-MEL-28 >7.68E−7  SK-MEL-5 2.14E−7 UACC-257 >7.68E−7  UACC-62 7.29E−7ovarian cancer IGROV1 >7.68E−7  OVCAR-3 >7.68E−7  OVCAR-4 >7.68E−7 OVCAR-8 1.90E−7 NCl/ADR-RES >7.68E−7  SK-OV-3 >7.68E−7  renal cancer786-0 6.03E−7 A498 4.38E−7 CAKI-1 5.16E−7 RXF 393 2.11E−7SN12C >7.68E−7  TK-10 >7.68E−7  UO-31 >7.68E−7  prostate cancer PC-33.06E−7 DU-145 4.62E−7 breast cancer MCF7 >7.68E−7  MDA-MB-231/ATCC1.18E−7 HS 578T 2.00E−7 BT-549 1.38E−7 T-47D >7.68E−7  MDA-MB-4681.79E−7

1-17. (canceled)
 18. A chimeric molecule comprising a lectin fused to atleast one heterologous polypeptide selected from the group consisting ofan epitope tag, an immunoglobulin, and a combination thereof.
 19. Thechimeric molecule of claim 18, wherein the lectin binds sialic acidthereby reducing tumor cell growth.
 20. The chimeric molecule of claim18, wherein the lectin is selected from a plant lectin, a viral lectin,a bacterial lectin, a fungi lectin, an insect lectin, and an animallectin.
 21. The chimeric molecule of claim 18, wherein the lectin is aplant lectin.
 22. The chimeric molecule of claim 21, wherein the plantis Maackia amurensis, Trichosanthes japonica, Viscum album, Artocarpusintergrifolia, or Sambucus nigra.
 23. The chimeric molecule of claim 18,wherein the lectin comprises SEQ ID NO:23.
 24. The chimeric molecule ofclaim 18, wherein the polypeptide is a polyhistidine epitope tag. 25.The chimeric molecule of claim 18, wherein the polypeptide comprises animmunoglobulin or a region thereof.
 26. The chimeric molecule of claim18, further comprising a detecting agent.
 27. The chimeric molecule ofclaim 26, wherein the detecting agent is a fluorescent moiety.
 28. Apharmaceutical composition comprising the chimeric molecule of claim 18and a pharmaceutically acceptable carrier.
 29. A kit comprising thechimeric molecule of claim
 18. 30. A method for reducing cancer cellgrowth comprising administering to a subject in need thereof aneffective amount of the chimeric molecule of claim
 18. 31. The method ofclaim 30, wherein the chimeric molecule is administered orally.
 32. Themethod of claim 30, wherein said cancer is selected from leukemia,non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovariancancer, renal cancer, prostate cancer and breast cancer.
 33. A method oftargeting Pdpn expressed on a cell comprising contacting said cell witha composition comprising the chimeric molecule of claim
 18. 34. A methodof targeting Pdpn expressed on a cell comprising: providing a cellculture comprising tumor cells and/or normal cells; subjecting saidcells to sufficient amount of an isolated lectin consisting of aminoacid sequence as set forth in the amino acid sequence set forth in theSEQ ID NO: 23; allowing sufficient amount of time for said lectin tobind cellular Pdpn thereby forming a lectin-Pdpn molecular complexcomprising at least a portion of the amino acid sequence set forth inthe SEQ ID No: 23 and wherein formation of said complex inhibits theactivity of Pdpn.
 35. The method of claim 34, further comprisingidentifying cells over-expressed with Pdpn by immunoassaying.
 36. Themethod of claim 35, wherein said immunoassaying is immunofluorescencemicroscopy.
 37. The method of claim 34, wherein said cancer is selectedfrom leukemia, non-small cell lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer and breastcancer.
 38. A method for inhibiting cancer cell growth comprisingidentifying cells over-expressed with Pdpn and contacting said cellswith an effective amount of lectin that binds sialic acid consisting ofan amino acid sequence as set forth in SEQ ID NO: 23, thereby reducingtumor cell growth.
 39. The method of claim 37, wherein said cancer isselected from skin cancer, head and neck cancer, leukemia, non-smallcell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer,renal cancer, prostate cancer and breast cancer.
 40. The method of claim37, wherein the cancer cell growth is inhibited by about 75% as comparedto a cancer cell not contacted with the lectin.
 41. A pharmaceuticalcomposition comprising an isolated lectin that binds sialic acid whereinthe lectin consist of the amino acid sequence as set forth in SEQ ID NO:23 in a mixture with a pharmaceutically acceptable carrier comprising adiluting agent, a flavor additive, a solvent, a lubricant, a suspensionagent, a binder, a preservative, a tablet-disintegrating substance or anencapsulating material.
 42. The pharmaceutical composition of claim 41,which is in a solid dosage form selected from powders, tablets, pills,capsules, suppositories, and dispersible granules.
 43. Thepharmaceutical composition of claim 42, further comprising at least twoor more pharmaceutically acceptable carriers selected from a dilutingagent, flavor additive, binder, and tablet-disintegrating substance. 44.The pharmaceutical composition of claim 41, which is in a liquid dosageform selected from a solution, a suspension, and an emulsion.
 45. Thepharmaceutical composition of claim 41, further comprising one or morepharmaceutically acceptable carriers selected from an artificialcoloring agent, a flavoring agent, a stabilizer, a buffer, a natural orartificial sweetener, a dispersing agent, a thickener, and a dissolvingagent.
 46. The pharmaceutical composition of claim 41, furthercomprising one or more pharmaceutically acceptable carriers selectedfrom magnesium carbonate, magnesium stearate, talc, sugar, lactose,pectin, dextrin, stiffeners, gelatins, tragacanth, methylcellulose, andsodium carboxymethylcellulose.